Chemical Standardization and Anti-Proliferative Activity of Ardisia elliptica Fruit against the HCT116 Human Colon Cancer Cell Line

Abstract

The present study is intended to carry out the chemical standardization and evaluation of the anti-proliferative activity of A. elliptica fruit extract. A. elliptica fruit powder was extracted with ethanol. The obtained extract was assessed for total phenolic content using the Folin-Ciocalteu method. Moreover, a simple, accurate, and precise reversed phase high-performance liquid chromatographic method was developed and validated to determine the embelin content of A. elliptica fruit extract. Then, the extract and embelin were investigated for their anti-proliferative effect against HCT-116 cells. Finally, the mechanisms of inhibition of the extract and embelin on the mRNA expression of pro-apoptotic genes Bad, Bax, and Caspase-8 and anti-apoptotic genes c-IAP1, Mcl-1, and XIAP were determined by real-time qRT-PCR. The phenolic content and embelin content of the extract were 5.20 ± 0.01 g of gallic acid equivalent per 100 g of dried fruit (g% GAE) and 5.57 ± 0.56 mg/g of extract, respectively. The extract and embelin showed strong anti-proliferative effects on HCT-116 cells with 50% inhibition concentration (IC₅₀) values of 19.16 ± 1.09 µg/mL and 25.93 ± 1.75 µg/mL, respectively. The A. elliptica extract exhibited a significant increase in the mRNA level of Bad, Bax, and Caspase-8 and a significant decrease in c-IAP1, Mcl-1, and XIAP. Embelin showed a significant decrease in Mcl-1 and XIAP.

Keywords: Ardisia elliptica; HCT-116; XIAP.; anti-proliferative; colon cancer; embelin; standardization.

Figure 1

Thin layer chromagography (TLC) chromatogram of Ardisia elliptica fruit extract; 1 = gallic acid, 2 = embelin, 3 = Ardisia elliptica fruit extract, adsorbent: silica gel GF254. Solvent system: 1 = acetate:glacial acetic acid:formic acid:hexane (15:2:2:10, v/v/v/v), 2 = ethyl acetate:toluene:formic acid (9:10:2, v/v/v). detection: A = UV 254 nm, B = UV 366 nm, and C = NP/PEG under UV 366 nm. Band identification system 1: gallic acid (Rf = 0.46), embelin (Rf = 0.62). Band identification system 2: gallic acid (Rf = 0.26), embelin (Rf = 0.46).

Figure 2

UV spectrum at 200–400 nm of standard embelin (A), HPLC chromatograms determined at 285 nm of standard embelin at a concentration of 20 µg/mL (RT = 3.66 min) (B), and HPLC chromatogram determined at 285 nm of A. elliptica fruit extract at a concentration of 1 mg/mL (retention time (RT) = 3.62 min) (C).

Figure 3

The anti-proliferative effects of A. elliptica fruit extract (A) and embelin (B) against HCT-116 cells after 48 h of incubation.

Figure 4

Effects of A. elliptica fruit extract and embelin on mRNA expressions of pro- and anti-apoptotic genes (A–F). Cells were treated with the vehicle (control), 10 µg/mL fruit extracts, or 20 µg/mL embelin for 12 h at 37 °C. After treatment, the total RNA was extracted from the cells, and the mRNA expression was analyzed using specific primers. The relative Bad (A), Bax (B), Caspase-8 (C), c-IAP1 (D), Mcl-1 (E), and XIAP (F) mRNA levels were quantified and shown as the mean ± SEM (N = 3). * p < 0.05 versus vehicle.

Figure 5

Schematic diagram illustrating the mechanisms of A. elliptica fruit extract (AE extract) and embelin on apoptotic pathway in HCT-116 cells.